Blastospore Production Research Articles

Dissolved oxygen levels affect dimorphic growth by the entomopathogenic fungus Isaria fumosorosea

The entomopathogenic fungus Isaria fumosorosea is capable of dimorphic growth (hyphal or yeast-like) in submerged culture. Using 250-mL baffled flasks, culture volumes of 50, 100, 150, and 200 mL were grown in a shaker incubator at 350 rpm and 28°C. Dissolved oxygen (DO) was continuously monitored using a non-invasive oxygen monitoring system. Culture volumes of 50 mL maintained DO concentrations above 10% throughout the 3-day growth period and accumulated biomass and produced blastospores more rapidly (1.2×109 blastospores mL−1 in 2 days) than the other culture volumes tested. Dissolved oxygen was depleted in culture volumes of 100, 150, and 200 mL after 20.5, 16.8, and 13.5 h, respectively. The DO in 150 and 200 mL cultures remained exhausted (<3%) throughout the growth period resulting in significantly lower blastospore yields and increased hyphal growth. These results were used to establish oxygen levels (>20% DO) for I. fumosorosea growth in 100-L bioreactors resulting in blastospore production (1.1×109 blastospores mL−1 in 2 days) comparable to highly aerated, low volume shake flask cultures. In addition, maintaining higher DO levels resulted in increased blastospore production by cultures of I. fumosorosea grown on low-cost nitrogen sources (cottonseed meal and soy flour) that previously elicited excessive hyphal growth. These studies showed that oxygen availability is essential for significant yeast-like growth by I. fumosorosea cultures and that continuous monitoring of oxygen concentrations in shake flask cultures can be used to establish aeration conditions for bioreactors.

Production of mycelium and blastospores of sp. in submerged culture

Hirsutella sp. was grown in four liquid media containing either casamino acids, corn steep liquor, collagen peptone or casein peptone. These media were inoculated with a 7 day-old culture of mycelia and blastospores of Hirsutella sp. and the cultures incubated with shaking at 250 rpm at 26°C. The media containing corn steep liquor, casamino acids or collagen peptone produced abundant mycelium, varying from 64 to 76.3 mg/ml at different fermentation days, whereas the medium containing casein peptone produced less biomass. Additionally, the casamino acids and collagen peptone media showed similar biomass values, as well as blastospore counts after 14 fermentation days, without significant differences between the two media. The highest number of blastospores produced was 3.8 × 10 7 blastospores/ml using medium with casamino acids. This medium, as well as that with collagen peptone gave suitable results for the preparation of an inoculum for the production of conidia in solid medium. Key words: Hirsutella , liquid media, fermentation, blastospores, microbial control, mass propagation.

Isolation and In Vitro Cultivation of Auriculoscypha anacardiicola D.A. Reid et Manim., an Insect-Associated and Potentially Medicinal Fungus from India

Auriculoscypha anacardiicola, an obligate insect-associate and a potential medicinal fungus, is isolated and studied in vitro. Suitable methods for isolation and cultivation of the fungus have been developed. Incubating spore deposits made to fall from basidiomata on tap water agar seems to be the best method for developing cultures. Successful isolations were also accomplished from infected coccids. Cultures could not be developed from single basidiospores and from tissues of the basidiomata. Although production of ballistospores and blastospores as well as germ tube formation were observed at the time of germination of basidiospores, budding blastospores alone produced mycelial cultures. Observations such as the inability of single basidiospores to germinate, emergence of mycelium from a spore deposit, and the apparent conjugation of yeast cells indicate that dikaryotization resulting from fusion of compatible yeast cells is essential for development of mycelium in A. anacardiicola. The fungus grew well on all complex media tested. It seems that a purely synthetic medium devoid of any growth factors cannot support the growth ofA. anacardiicola and yeast extract seems to provide the required growth factors.

English

&nbsp; There are many advantages to using liquid cultures for the production of blastospores. These include mainly the processes of scale up which are relatively easy, as well as the control of parameters such as temperature, aeration and pH. In this work, we evaluated the production of&nbsp;Paecilomyces fumosoroseus&nbsp;blastospores using a low-cost liquid culture medium in a fermenter in comparison to a medium commonly used for this purpose, with regard to yield and viability of blastospores. The two media contained the same concentration of glucose but differed in N source (M1 containing casamino acids and M2 provided with collagen peptone and yeast extract). Starting with an inoculum of 1x106&nbsp;blastospores/ml, M2 medium produced 2x1010&nbsp;blastospores/ml after incubation for 72 h at 520 rev/min agitation and 1 v/v/m (volume air/volume&nbsp;liquid.min) aeration, while only 2.4 x 108/ml were produced with M1. In addition, the microorganisms in medium M1 grew more slowly during log phase and reached an earlier plateau at 36 h fermentation. The medium containing collagen peptone and yeast extract is an excellent alternative for the production of&nbsp;P.&nbsp;fumosoroseus&nbsp;blastospores, providing lower cost, higher yield and shorter propagation time, but formulation does need to be improved. &nbsp; Key words:&nbsp;Collagen peptone, low cost, short times of propagation, fed-batch.

The MAD1 Adhesin of Metarhizium anisopliae Links Adhesion with Blastospore Production and Virulence to Insects, and the MAD2 Adhesin Enables Attachment to Plants

Metarhizium anisopliae is a fungus of considerable metabolic and ecological versatility, being a potent insect pathogen that can also colonize plant roots. The mechanistic details of these interactions are unresolved. We provide evidence that M. anisopliae adheres to insects and plants using two different proteins, MAD1 and MAD2, that are differentially induced in insect hemolymph and plant root exudates, respectively, and produce regional localization of adhesive conidial surfaces. Expression of Mad1 in Saccharomyces cerevisiae allowed this yeast to adhere to insect cuticle. Expression of Mad2 caused yeast cells to adhere to a plant surface. Our study demonstrated that as well as allowing adhesion to insects, MAD1 at the surface of M. anisopliae conidia or blastospores is required to orientate the cytoskeleton and stimulate the expression of genes involved in the cell cycle. Consequently, the disruption of Mad1 in M. anisopliae delayed germination, suppressed blastospore formation, and greatly reduced virulence to caterpillars. The disruption of Mad2 blocked the adhesion of M. anisopliae to plant epidermis but had no effects on fungal differentiation and entomopathogenicity. Thus, regulation, localization, and specificity control the functional distinction between Mad1 and Mad2 and enable M. anisopliae cells to adapt their adhesive properties to different habitats.

Characterisation of optimum cultural environmental conditions for the production of high numbers of Metarhizium anisopliae blastospores with enhanced ecological fitness

The aim of this study was the production of high numbers of M. anisopliae blastospores with enhanced germination efficiency under conditions of water-stress (ecological fitness). Different nitrogen sources and concentrations were screened for their ability to induce blastospore production while keeping pH and water activity (a w) at fixed levels (6.8, and 0.98 a w, respectively). After optimum nitrogen status was determined (cornsteep solid (CS) + yeast extract (YE); or cottonseed flour (CF) + YE), the effect of interaction of nitrogen source, pH (3.5, 5, 6.8, 8, 9 and 10) and solutes for a w adjustment (KCl, NaCl, PEG 200) on blastospore production, endogenous polyol content (glycerol, erythritol, arabitol and mannitol) and total protein, were determined. For both ionic (NaCl, KCl) and non-ionic solutes (PEG 200), optimum blastospore production (between 4×107 and 2×108 blastospores ml−1) and growth occurred in the pH range 6.8–8, with the CF + YE nitrogen profile giving higher yields than CS + YE. Optimum conditions for high erythritol and total protein endogenous concentrations (40.37–73.44 and 14.33–18.90 mg g−1 fresh weight, respectively) occurred between pH 6.8 and 8 by ionic aw modification (KCl, NaCl) and with the CF + YE nitrogen profile. Germination of blastospores produced under these cultural conditions was between 62 and 89% under conditions of water-stress (0.96 a w). On the other hand, blastospores with lower amounts of erythritol and total protein content had decreased germination (8–67%). These results could have significant implications for developing a liquid fermentation medium for the production of high numbers of fungal propagules with enhanced efficacy under non-optimum environmental conditions.

Impact of carbon and nitrogen nutrition on the quality, yield and composition of blastospores of the bioinsecticidal fungus Paecilomyces fumosoroseus

The impact of growing cultures of Paecilomyces fumosoroseus in liquid media containing four combinations of glucose and casamino acids (8 g l(-1) or 80 g l(-1) glucose, 1.32 g l(-1) or 13.2 g l(-1) casamino acids) was evaluated, based on blastospore production, germination rate, viability after freeze-drying and short-term storage stability. When blastospores were produced using a high casamino acid concentration, blastospore yields and germination rates were significantly higher (13.2-18.5 x 10(7) blastospores ml(-1), 50-60% germination after 4 h), compared to cultures grown in media containing lower casamino acid concentrations (0.4-2.3 x 10(7) blastospores ml(-1), 10-20% germination after 4 h). Chemical analyses of blastospore composition showed that accelerated blastospore germination may be related to increased proteinaceous reserves rather than to glycogen or lipid accumulation. Tolerance to freeze-drying by blastospores suspended in spent medium was enhanced by a high initial casamino acid concentration in the culture medium (75% survival) and by the residual glucose concentrations in the spent medium. Under the conditions of this study, the storage stability of blastospores of P. fumosoroseus was unaffected by the nutritional condition in which they were produced.

Impact of water-stress and washing treatments on production, synthesis and retention of endogenous sugar alcohols and germinability of Metarhizium anisopliae blastospores

The entomopathogenic fungus Metarhizium anisopliae was physiologically manipulated by culturing in liquid culture under a range of water-stress conditions (0.98, 0.97 and 0.96 water activity, a(w)) imposed by either PEG 200, KCl or NaCl at 25 degrees C to optimise blastospore production and quality in terms of accumulation of endogenous compatible solutes and germinability. Optimum spore yield occurred at 0.98a(w), when PEG 200 was the solute. Blastospores produced under different water-stress levels were subjected to hypo-osmotic and iso-osmotic washing treatments before quantification of endogenous sugar alcohols (polyols) and germinability studies. Endogenous amounts of the low molecular weight polyol erythritol were greatly affected by culture age, water-stress and hypotonic or isotonic washing treatment. Endogenous erythritol in blastospores produced under 0.98 and 0.97a(w) increased with culture age. Osmoprotection (washing with isotonic PEG 200 solution) of blastospores produced under all water-stress conditions resulted in retention of higher amounts of erythritol. Increased endogenous retention of the high molecular weight polyol mannitol was also observed with an isotonic washing treatment but only in blastospores produced under intermediate water-stress (0.98 and 0.97a(w)). Germination of all treatments was > 90 % on a medium with freely available water. Under water-stress conditions (0.96a(w)) only 10 % of unmodified blastospores washed with water germinated. However, those grown under modified water availability conditions (0.98a(w)) and washed with isotonic solutions had significantly improved germination (38-90 %). Blastospores produced at < or = 0.97a(w) washed with isotonic solutions had decreased germination. These results could have significant implications for conserving ecophysiological quality of fungal biocontrol propagules during harvesting and have an impact on storage and formulation.

Influence of nutrient, pH and dissolved oxygen on the production of Metarhizium flavoviride Mf189 blastospores in submerged batch culture

The influence of different parameters on the sporulation of Metarhizium flavoviride was studied during submerged cultures in shake flasks and in 5 l bioreactors. The screening in shake flasks of several carbon and nitrogen sources allowed the definition of an optimal medium, based on sucrose and brewer's yeast with a C/N ratio of 1.6. With this medium, a production of 5.4 × 10 8 blastospores per ml (Bspores ml −1) was obtained after 169 h of cultivation. The influence of pH and pO 2 was independently studied in 5 l working volume bioreactors using the optimal medium. The best production was obtained with pH and pO 2 regulated respectively to 7 and 100%. Finally, when the culture was grown under optimized conditions, the blastospores concentration increased 16-fold, with 1.1 × 10 9 Bspores ml −1 obtained after 144 h cultivation. This represents a gain of productivity of about 4.8 times.

Liquid-culture production of blastospores of the bioinsecticidal fungus Paecilomyces fumosoroseus using portable fermentation equipment.

The production of fungal spores using on-site, non-sterile, portable fermentation equipment is technically constrained. Very little information is available on the production requirements, such as medium concentration, inoculum stabilization, required fermentation times, and maintenance of axenic growth. In this study, we developed a two-part, liquid concentrate of the production medium that remains stable and soluble at room temperature. We also examined inoculum stability and showed that freeze- or air-dried blastospore preparations were stable for 7 days after rehydration when stored at 4 degrees C. The use of a low-pH (pH 4), relatively rich complex medium provided a growth environment deleterious to bacterial growth yet conducive to rapid sporulation by Paecilomyces fumosoroseus. High concentrations of blastospores (7.9 x 10(8)/ml) of P. fumosoroseus were produced in a 40-h fermentation with very low levels of bacterial contamination when the fermentor was charged with a blastospore production medium with a starting pH of 4 and inoculated with blastospore concentrations greater than 1 x 10(6) spores/ml. These studies demonstrate that the use of disinfected, portable fermentation equipment has potential for on-site production of high concentrations of blastospores of the bioinsecticidal fungus P. fumosoroseus.

Biomass and Blastospore Production in Beauveria bassiana (Bais.) Vuill. as Influenced by Media Components

Beauveria bassiana , an entomopathogenic fungus, was grown under submerged condition using six different media. When different supplements were studied, 2 percent polyethylene glycol (PEG) favoured both higher biomass (102mg/mI) and blastospore (14 × 10 8 ) yield, while chitin (0.2%), favoured only higher biomass (101mg/ml) turn over.

Drying and formulation of blastospores of Paecilomyces fumosoroseus (Hyphomycetes) produced in two different liquid media

Formulation matrices can play an important role in improving the storage survival and biocontrol efficacy of microorganisms used for the control of pest insects. In this study, liquid culture-produced blastospores of the entomopathogenic fungus Paecilomyces fumosoroseus were formulated with different inert and organic materials prior to air-drying. Paecilomyces fumosoroseus blastospores were produced in two different liquid media, a basal salts medium supplemented with Casamino acids and glucose (LM1) and a medium containing peptone of collagen and glucose (LM2). Blastospores produced in the two test media were formulated with various supports. The formulation supports were cornstarch, rice flour, talc powders, Mexican lime, calcined kaolin clay, and diatomaceous earth. Several of the supports were tested at different concentrations. The initial and long-term (after storage at 4 and 28 °C) survival of the formulated, air-dried blastospores were evaluated. Initial blastospore viabilities were affected by the formulation material and by the blastospore production medium. Medium composition, drying support and storage temperature had an impact on the long-term survival of the blastospores. Under the conditions of the study, LM1 produced higher concentrations of blastospores that not only survived drying better than blastospores produced in LM2 but also maintained viability longer during storage in the formulation supports tested. The nature of the drying supports was shown to have a significant impact on the storage stability of all blastospores, particularly those produced in LM1. Under the production, drying and storage conditions used in the study, calcined kaolin clay formulations stored at 4 °C had the best storage stability. In all formulations tested, spore survival over time was reduced for blastospore formulations stored at 28 °C rather than 4 °C.

Influence of culture conditions on production and freeze-drying tolerance of Paecilomyces fumosoroseus blastospores.

With the goal of developing a defined medium for the production of desiccation-tolerant blastospores of the bioinsecticidal fungus Paecilomyces fumosoroseus, we evaluated the impact of various media components such as amino acids, carbohydrates, trace metals and vitamins on hyphal growth and sporulation of P. fumosoroseus cultures and on the freeze-drying tolerance of blastospores produced under these conditions. A comparison of 13 amino acids as sole nitrogen sources showed that glutamate, aspartate, glycine and arginine supported biomass accumulations (12-16 mg ml-1) and blastospore yields (6-11 x 108 blastospores ml-1) comparable to our standard production medium which contains casamino acids as the nitrogen source. Using glutamate as the sole nitrogen source, tests with various carbohydrates showed that P. fumosoroseus grew best on glucose (18.8 mg biomass ml-1) but produced similar blastospore concentrations (7.3-11.0 x 108) when grown with glucose, glycerol, fructose or sucrose. P. fumosoroseus cultures grown in media with sodium citrate or galactose as the sole carbohydrate produced lower blastospore concentrations but more-desiccation-tolerant spores. Zinc was the only trace metal tested that was required for optimal growth and sporulation. In a defined medium with glutamate as the nitrogen source, vitamins were unnecessary for P. fumosoroseus growth or sporulation. When blastospores were freeze-dried in the absence of a suspension medium, residual glucose (>2.5% w/v) was required for enhanced spore survival. Thus, a defined medium containing basal salts, glucose, glutamate and zinc can be used to produce optimal concentrations of desiccation-tolerant blastospores of P. fumosoroseus.

Effect of Additives on the Production, Viability and Virulence of Blastospores of Metarhizium anisopliae

Studies on the blastospore production of Metarhizium anisopliae var. anisopliae were conducted in Adamek's medium used as a standard, enriched with lecithin, collagen, lactic acid or polyethylene glycol 200 (PEG 200) to increase spore yield and suppress mycelial pellet formation. The addition of 5% lecithin resulted in a significant 10-fold increase in spore yield up to 1.9 108 blastospores/ml compared with 1.9 107 spores/ml in the standard medium. Collagen (3%) increased the number of blastospores 3.7-fold, and lactic acid (1.5%) two-fold. A reduction of mycelial pellet formation in favour of spore production was noted with each additive. The viability of blastospores at 40IC from media with lecithin, collagen and lactic acid suspended in 25% Ringer's solution was comparable to that of spores produced in the standard medium. Striking differences were noticed in the viability of spores produced with 5% PEG 200 in standard medium. The half-life of blastospores produced in standard medium suspended in sunflower oil was 33.6 h and that of 5% PEG 200 spores only 25.2 h. In bioassays, the virulence of spores produced in standard medium to which 3% lecithin, 3% collagen, 1.5% lactic acid or 5% PEG 200 had been added was tested against third-instar nymphs of Locusta migratoria migratorioides (R. & F.). The median lethal time and the mortality of L. migratoria achieved with blastospores produced with 3% lecithin (5.7 days, 99%) was comparable to that of blastospores from standard medium (5.1 days, 98%). The virulence of blastospores from all other media with additives was significantly reduced.

Liquid culture production of desiccation tolerant blastospores of the bioinsecticidal fungus Paecilomyces fumosoroseus

Liquid media with differing carbon concentrations and carbon-to-nitrogen ratios were tested for production of desiccation tolerant blastospores of Paecilomyces fumosoroseus. While all media tested supported sporulation in submerged culture, high blastospore concentrations (5·8 × 108) spores ml−) were produced in media containing 80 g glucose l− and 13·2 g Casamino acids l− (MS medium) and a significantly higher percentage (79%) of these blastospores survived air drying. Media containing glucose concentrations greater than 20 g l − and Casamino acid concentrations between 13·2 and 40 g l− supported maximal production of desiccation tolerant blastospores. All 23 isolates of P. fumosoroseus grown in MS media produced high concentrations of desiccation tolerant blastospores. When stored at 4 °C, more than 60% of the lyophilized blastospores produced in MS medium were still viable after 7 months storage while less than 25% of the air-dried blastospores survived after 90 d storage. Standard whitefly bioassays were performed to compare air-dried blastospores of P. fumosoroseus ARSEF 4491 with solid substrate-produced conidia of Beauveria bassiana ARSEF 252. Air-dried blastospores of P. fumosoroseus gave LD50s of 60 and 113 blastospores mm− for the silverleaf whitefly (Bemisia argentifolii) in two separate bioassays with potency ratios (LD50 B. bassiana/LD50 P. fumosoroseus)of 3·9 and 3·8, respectively. These results have demonstrated that high concentrations of blastospores of P. fumosoroseus can be rapidly produced in liquid culture, remain viable following drying, and infect and kill silverleaf whitefly.

Effects of naftifine and terbinafine, two allylamine antifungal drugs, on selected functions of human polymorphonuclear leukocytes

Many antimycotic agents negatively affect the natural immune response. Typically, these drugs impair polymorphonuclear leukocyte (PMN) production of superoxide anion, chemotaxis, or the killing of pathogens. Allylamines are a new class of antimycotic compounds with a new mechanism of antifungal action, i.e., inhibition of the fungal squalene epoxidase. The trial that we describe aimed to evaluate the effects of two allylamines, terbinafine and naftifine, on selected functions of PMNs, i.e., superoxide anion production, chemotaxis, and killing of Candida albicans blastospores. Terbinafine and naftifine on their own did not affect superoxide anion production when they were added to PMNs. When PMNs were preincubated with allylamines and were then stimulated by N-formyl-Met-Leu-Phe or phorbol 12-myristate 13-acetate, superoxide anion production was increased (priming effect). Since intracellular free calcium (Ca2+i) is involved in the control of superoxide anion production, we evaluated the effects of the allylamines on the Ca2+i concentration ([Ca2+]i). In the presence of terbinafine or naftifine, the [Ca2+]i increased in a dose-dependent manner; the source of Ca2+i was not extracellular since it was not affected by extracellular calcium chelation with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. In the presence of terbinafine or naftifine, chemotaxis of PMNs was not impaired. Terbinafine and naftifine slightly but significantly increased the killing of C. albicans blastospores (P < 0.05 at 10 and 100 microM). In conclusion, in contrast to imidazole-like drugs, the allylamine antimycotic compounds terbinafine and naftifine enhance selected functions of PMNs.

Production, formulation and application of the entomopathogenic fungusBeauveria bassianafor insect control: current status

This review summarizes the progress and achievements made in the last decade in mass production formulation and application technology of the entomopathogenic fungus Beauveria bassiana. Reports published on relevant research from Belgium, Canada, China, Cuba, Czechoslovakia (former), France, Germany, Great Britain, Philippines, Poland, Switzerland, USA and USSR (former) regarding this topic have been covered. Much of the non‐English language literature, particularly that from Eastern European and Chinese sources, has not been translated and is inaccessible to most English or other western language readers. We have done this translation and through this review provide technological details about mass production of B. bassiana in China. Various aspects of B. bassiana growth, substrate use, production of mycelia, conidiospore and blastospores, process technologies associated with separation, drying and milling, formulation, storage and ‘shelf‐life’, and field efficacy are reviewed. Data are presented on: a m...

Production of blastospores by three strains of metarhizium anisopliae (metch.) sorokin in submerged culture

Studies on blastospore production in different liquid media were conducted with three strains of Metarhizium anisopliae var. anisopliae (M. a.) derived from various countries (M. a. 43: Austria, M. a. 57: Brazil, M. a. 97: Philippines). Variation of six fermentation parameters (cornsteep products, carbohydrates, pH values, temperature, Tween 80, and polyethyleneglycol (PEG) 200) disclosed that the three strains of M. anisopliae differed in their growth pattern and physiology. In standard medium and in all tests, M. a. 57 produced the highest number of blastospores invariably amounting to > 108 per ml, while mycelial pellets were never formed. The preferred carbohydrates were glucose and fructose. Blastospore production of M. a. 43 was increased by growth at 30°C, at pH 6.5 or by addition of 5% PEG 200. However, it was impaired by different concentrations of Tween 80 or higher concentrations of PEG 200 (10–15%). M. a. 97 produced most blastospores at 30°C, and the strain preferred basic (pH 8.0) as well as...

Comparison of sporulation of Paecilomyces farinosus and Beauveria bassiana in batch and fed-batch culture

The entomogenous fungi, Paecilomyces farinosus and Beauveria bassiana, were grown in batch and in glucose- and ammonium-limited fed-batch culture. In batch cultures grown on complex or minimal media, P. farinosus and B. bassiana produced blastospores in the exponential and deceleration phases of growth and B. bassiana, but not P. farinosus, also formed spores during the stationary phase. In both glucose- and ammonium-limited fed-batch cultures (complex and minimal media for glucose, minimal medium for ammonium), blastospore concentration in P. farinosus, but not B. bassiana, was inversely related to dilution rate. However, compared with batch cultures grown on complex medium, only glucose-limited fed-batch cultures of P. farinosus grown on minimal medium gave enhanced production of blastospores. Unbalanced growth occurred in ammonium-limited fed-batch cultures of both fungi.

Effects of water activity on morphology, growth and blastospore production of Metarhizium anisopliae, Beauveria bassiana and Paecilomyces farinosus in batch and fed-batch culture

Metarhizium anisopliae, Beauveria bassiana and Paecilomyces farinosus were grown in submerged liquid culture on glucose- and polyethylene glycol (PEG)-adjusted media of different initial water activities. The initial glucose or PEG concentration of the medium affected the morphology, biomass and blastospore production of M. anisopliae and P. farinosus in shake flask culture. At 80 mg glucose ml−1 or 0·15 M PEG (initial water activity (aw), 0·980), M. anisopliae grew filamentously, but at 10 mg glucose ml−1 or in the absence of PEG (initial aw, 0·986), pellet growth occurred. Similarly, less mycelial aggregation occurred in cultures of P. farinosus grown at low (aw, 0·982) than at high (aw, 0·990) initial water activities. Conditions which favoured a filamentous morphology were correlated with increased blastospore production. Blastospore yield of fed-batch cultures of B. bassiana was increased when the water activity of the nutrient feed was reduced by the incorporation of 2·4 M PEG.